This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract: Ricin is a highly potent and rapidly acting biological toxin which has been employed for biologic warfare. An effective strategy to protect threatened populations against biological attack by ricin is to generate vaccines comprising a modified ricin. RiVax is a novel genetically modified ricin vaccine candidate that has shown promising but not optimal results in nonhuman primate models when parenterally administered. The efficacy of RiVax may be significantly improved by altering the site and manner of its delivery. First, nasal vaccination provides a needle-free method of immunization that has the potential to induce antigen-specific systemic IgG as well as mucosal IgA. Second, heterologous prime/boost immunization regimens often induce immune responses that are superior to those induced by homologous prime/boost immunization. We hypothesized that the use of a novel adjuvant (Mastoparan-7;MP-7) and a novel nasal delivery method will enhance the immunogenicity of the nasally delivered RiVax vaccine and provide a needle-free method of immunization that when combined with a parenteral RiVax priming immunization, will induce antibodies in vaccinated animals. Our specific aim is to determine if a heterologous prime/boost immunization strategy utilizing a parenteral prime and a nasal boost with RiVax is superior to parenteral immunization for its ability to induce serum ricin-neutralizing antibodies. A group of rhesus macaques (M. mulatta) (n=6) were heterologously vaccinated by prime immunizing intramuscularly (IM) with RiVax and then boosted twice with ricin vaccine (protein only) admixed with Mastoparan-7 and administered intranasally (IN) 30 days apart. Other groups were primed and boosted with RiVax admixed with aluminum hydroxide by the same schedule using only IM injection as the vaccination route or sham vaccinated with saline. Serum immune response among the groups receiving vaccine were comparable regardless of route, with endpoint IgG titer ranging from 1:4,500 to 1:10,500 in the animals receiving vaccine heterologously or solely by IM injection, respectively. Neutralizing capability of serum antibodies, as determined by an in vitro cytotoxicity assay, showed little to no activity in the animals receiving the vaccine heterologously, whereas the group receiving the vaccine IM resulted in neutralizing titers up to 1:400. Secretory IgA analysis of bronchoalevolar lavage and buccal swabs is in process. Approximately 45 days after the 2nd boost, all animals were challenged with ricin toxin by aerosol at a dose equivalent to multiple (2-5) LD^50s. All animals in the sham-vaccinated group succumbed to intoxication approximately +45-50 hours postexposure. One of the six animals (1/6;16%) in the IM-vaccinated group survived challenge. Four of the six animals in the heterologous vaccinated group survived challenge (4/6;66%). Results indicate that the heterologous route of vaccine administration paired with the use of MP-7 as a mucosal adjuvant proved more efficacious than traditional vaccine administration, although serum IgG levels of a-ricin neutralizing antibodies was not predictive of this response. Increased survival in the IN vaccinated group may be due to generation of secretory IgA that neutralized toxin at the site of entry rather than in the periphery of the animal, thereby reducing the toxic effects that lead to vascular leak syndrome.